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Biomedical subjects

Steven P Gross

Publications and source records attributed to Steven P Gross.

18 recordsLinked to original sources
PubMed2006

Guidance of bidirectional motor complexes by mRNA cargoes through control of dynein number and activity.

During asymmetric cytoplasmic mRNA transport, cis-acting localization signals are widely assumed to tether a specific subset of transcripts to motor complexes that have intrinsic directionality. Here we provide evidence that mRNA transcripts control their sorting by regulating the relative activities of opposing motors on microtubules. We show in Drosophila embryos that all mRNAs undergo bidirectional transport on microtubules and that cis-acting elements produce a range of polarized transcript distributions by regulating the frequency, velocity, and duration of minus-end-directed runs. Increased minus-end motility is dependent on the dosage of RNA elements and the proteins Egalitarian (Egl) and Bicaudal-D (BicD). We show that these proteins, together with the dynein motor, are recruited differentially to different RNA signals. Cytoplasmic transfer experiments reveal that, once assembled, cargo/motor complexes are insensitive to reduced cytoplasmic levels of transport proteins. Thus, the concentration of these proteins is only critical at the onset of transport. This work suggests that the architecture of RNA elements, through Egl and BicD, regulates directional transport by controlling the relative numbers of opposite polarity motors assembled. Our data raise the possibility that recruitment of different numbers of motors and regulatory proteins is a general strategy by which microtubule-based cargoes control their sorting.

Animals↗
PubMed2006

The Herpesvirus capsid surface protein, VP26, and the majority of the tegument proteins are dispensable for capsid transport toward the nucleus.

Upon entering a cell, alphaherpesvirus capsids are transported toward the minus ends of microtubules and ultimately deposit virus DNA within the host nucleus. The virus proteins that mediate this centripetal transport are unknown but are expected to be either viral tegument proteins, which are a group of capsid-associated proteins, or a surface component of the capsid itself. Starting with derivatives of pseudorabies virus that encode a fluorescent protein fused to a structural component of the virus, we have made a collection of 12 mutant viruses that lack either the VP26 capsid protein or an individual tegument protein. Using live-cell fluorescence microscopy, we tracked individual virus particles in axons following infection of primary sensory neurons. Quantitative analysis of the VP26-null virus indicates that this protein plays no observable role in capsid transport. Furthermore, viruses lacking tegument proteins that are nonessential for virus propagation in cell culture were also competent for axonal transport. These results indicate that a protein essential for viral propagation mediates transport of the capsid to the nucleus.

Animals↗
PubMed2005

Monte Carlo modeling of single-molecule cytoplasmic dynein.

Molecular motors are responsible for active transport and organization in the cell, underlying an enormous number of crucial biological processes. Dynein is more complicated in its structure and function than other motors. Recent experiments have found that, unlike other motors, dynein can take different size steps along microtubules depending on load and ATP concentration. We use Monte Carlo simulations to model the molecular motor function of cytoplasmic dynein at the single-molecule level. The theory relates dynein's enzymatic properties to its mechanical force production. Our simulations reproduce the main features of recent single-molecule experiments that found a discrete distribution of dynein step sizes, depending on load and ATP concentration. The model reproduces the large steps found experimentally under high ATP and no load by assuming that the ATP binding affinities at the secondary sites decrease as the number of ATP bound to these sites increases. Additionally, to capture the essential features of the step-size distribution at very low ATP concentration and no load, the ATP hydrolysis of the primary site must be dramatically reduced when none of the secondary sites have ATP bound to them. We make testable predictions that should guide future experiments related to dynein function.

Adenosine Triphosphate↗
PubMed2005

Regulation of lipid-droplet transport by the perilipin homolog LSD2.

BACKGROUND: Motor-driven transport along microtubules is a primary mechanism for moving and positioning organelles. How such transport is regulated remains poorly understood. For lipid droplets in Drosophila embryos, three distinct phases of transport can be distinguished. To identify factors regulating this transport, we biochemically purified droplets from individual phases and used 2D gel analysis to search for proteins whose amount on droplets changes as motion changes. RESULTS: By mass spectrometry, we identified one such protein as LSD2. Similar to its mammalian counterpart Perilipin, LSD2 is responsible for regulating lipid homeostasis. Using specific antibodies, we confirmed that LSD2 is present on embryonic lipid droplets. We find that lack of LSD2 causes a specific transport defect: Droplet distribution fails to undergo the dramatic changes characteristic of the wild-type. This defect is not due to a complete failure of the core transport machinery--individual droplets still move bidirectionally along microtubules with approximately normal velocities and kinetics. Rather, detailed biophysical analysis suggests that developmental control of droplet motion is lost. We show that LSD2 is multiply phosphorylated in a developmentally controlled manner. LSD2 phosphorylation depends on the transacting signal Halo, and LSD2 can physically interact with the lipid-droplet-associated coordinator Klar, identifying LSD2 as a central player in the mechanisms that control droplet motion. CONCLUSIONS: LSD2 appears to represent a new class of regulators, a protein that transduces regulatory signals to a separable core motor machinery. In addition, the demonstration that LSD2 regulates both transport and lipid metabolism suggests a link between lipid-droplet motion and lipid homeostasis.

Animals↗
PubMed2005

Neutrophil migration in opposing chemoattractant gradients using microfluidic chemotaxis devices.

Neutrophils migrating in tissue respond to complex overlapping signals generated by a variety of chemotactic factors (CFs). Previous studies suggested a hierarchy between bacteria-derived CFs and host-derived CFs but could not differentiate neutrophil response to potentially equal host-derived CFs (IL-8 and LTB4). This paper reports neutrophil migration in conflicting gradients of IL-8 and LTB4 using a microfluidic chemotaxis device that can generate stable and well-defined gradients. We quantitatively characterized the movement of cells from time-lapse images. Neutrophils migrate more efficiently toward single IL-8 gradients than single LTB4 gradients as measured by the effective chemotactic index (ECI). In opposing gradients of IL-8 and LTB4, neutrophils show obvious chemotaxis toward a distant gradient, consistent with previous reports. When an opposing gradient of LTB4 is present, neutrophils show less effective chemotaxis toward IL-8 than when they are in a gradient of IL-8 alone. In contrast, the chemotactic response of neutrophils to LTB4 is not reduced in opposing gradients as compared to that in a single LTB4 gradient. These results indicate that the presence of one host-derived CF modifies the response of neutrophils to a second CF suggesting a subtle hierarchy between them.

Cell Culture Techniques↗
PubMed2005

Tracking single particles: a user-friendly quantitative evaluation.

As our knowledge of biological processes advances, we are increasingly aware that cells actively position sub-cellular organelles and other constituents to control a wide range of biological processes. Many studies quantify the position and motion of, for example, fluorescently labeled proteins, protein aggregates, mRNA particles or virus particles. Both differential interference contrast (DIC) and fluorescence microscopy can visualize vesicles, nuclei or other small organelles moving inside cells. While such studies are increasingly important, there has been no complete analysis of the different tracking methods in use, especially from the practical point of view. Here we investigate these methods and clarify how well different algorithms work and also which factors play a role in assessing how accurately the position of an object can be determined. Specifically, we consider how ultimate performance is affected by magnification, by camera type (analog versus digital), by recording medium (VHS and SVHS tape versus direct tracking from camera), by image compression, by type of imaging used (fluorescence versus DIC images) and by a variety of sources of noise. We show that most methods are capable of nanometer scale accuracy under realistic conditions; tracking accuracy decreases with increasing noise. Surprisingly, accuracy is found to be insensitive to the numerical aperture, but, as expected, it scales with magnification, with higher magnification yielding improved accuracy (within limits of signal-to-noise). When noise is present at reasonable levels, the effect of image compression is in most cases small. Finally, we provide a free, robust implementation of a tracking algorithm that is easily downloaded and installed.

Algorithms↗
PubMed2004

Molecular motors: strategies to get along.

The majority of active transport in the cell is driven by three classes of molecular motors: the kinesin and dynein families that move toward the plus-end and minus-end of microtubules, respectively, and the unconventional myosin motors that move along actin filaments. Each class of motor has different properties, but in the cell they often function together. In this review we summarize what is known about their single-molecule properties and the possibilities for regulation of such properties. In view of new results on cytoplasmic dynein, we attempt to rationalize how these different classes of motors might work together as part of the intracellular transport machinery. We propose that kinesin and myosin are robust and highly efficient transporters, but with somewhat limited room for regulation of function. Because cytoplasmic dynein is less efficient and robust, to achieve function comparable to the other motors it requires a number of accessory proteins as well as multiple dyneins functioning together. This necessity for additional factors, as well as dynein's inherent complexity, in principle allows for greatly increased control of function by taking the factors away either singly or in combination. Thus, dynein's contribution relative to the other motors can be dynamically tuned, allowing the motors to function together differently in a variety of situations.

Biological Transport↗
PubMed2004

Intracellular actin-based transport: how far you go depends on how often you switch.

Intracellular molecular motor-driven transport is essential for such diverse processes as mitosis, neuronal function, and mitochondrial transport. Whereas there have been in vitro studies of how motors function at the single-molecule level, and in vivo studies of the structure of filamentary networks, studies of how the motors effectively use the networks for transportation have been lacking. We investigate how the combined system of myosin-V motors plus actin filaments is used to transport pigment granules in Xenopus melanophores. Experimentally, we characterize both the actin filament network, and how this transport is altered in response to external signals. We then develop a theoretical formalism to explain these changes. We show that cells regulate transport by controlling how often granules switch from one filament to another, rather than by altering individual motor activity at the single-molecule level, or by relying on structural changes in the network.

Actins↗
PubMed2004

Effective neutrophil chemotaxis is strongly influenced by mean IL-8 concentration.

Neutrophils need to correctly interpret gradients of chemotactic factors (CFs) such as interleukin 8 (IL-8) to migrate to the site of infection and perform immune functions. Because diffusion-based chemotaxis assays used in previous studies suffer from temporally changing gradients, it is difficult to distinguish the influence of CF gradient steepness from mean CF concentration on chemotaxis. To better understand the roles of mean CF concentration and CF gradient steepness, we developed a microfluidic device that can maintain stable IL-8 gradients. We report that the random motility of neutrophils is a biphasic function of IL-8 concentration and its magnitude plays a decisive role in effective chemotaxis, a quantitative measure of migration. We show that the concentrations for the optimum chemotaxis in linear IL-8 gradients and for the maximum random motility in uniform IL-8 coincide. In contrast, we find that the steepness of IL-8 gradients has no significant effect on effective chemotaxis.

Chemotaxis, Leukocyte↗
PubMed2004

Cytoplasmic dynein functions as a gear in response to load.

Cytoskeletal molecular motors belonging to the kinesin and dynein families transport cargos (for example, messenger RNA, endosomes, virus) on polymerized linear structures called microtubules in the cell. These 'nanomachines' use energy obtained from ATP hydrolysis to generate force, and move in a step-like manner on microtubules. Dynein has a complex and fundamentally different structure from other motor families. Thus, understanding dynein's force generation can yield new insight into the architecture and function of nanomachines. Here, we use an optical trap to quantify motion of polystyrene beads driven along microtubules by single cytoplasmic dynein motors. Under no load, dynein moves predominantly with a mixture of 24-nm and 32-nm steps. When moving against load applied by an optical trap, dynein can decrease step size to 8 nm and produce force up to 1.1 pN. This correlation between step size and force production is consistent with a molecular gear mechanism. The ability to take smaller but more powerful strokes under load--that is, to shift gears--depends on the availability of ATP. We propose a model whereby the gear is downshifted through load-induced binding of ATP at secondary sites in the dynein head.

Adenosine Triphosphate↗
PubMed2004

Hither and yon: a review of bi-directional microtubule-based transport.

Active transport is critical for cellular organization and function, and impaired transport has been linked to diseases such as neuronal degeneration. Much long distance transport in cells uses opposite polarity molecular motors of the kinesin and dynein families to move cargos along microtubules. It is increasingly clear that many cargos are moved by both sets of motors, and frequently reverse course. This review compares this bi-directional transport to the more well studied uni-directional transport. It discusses some bi-directionally moving cargos, and critically evaluates three different physical models for how such transport might occur. It then considers the evidence for the number of active motors per cargo, and how the net or average direction of transport might be controlled. The likelihood of a complex linking the activities of kinesin and dynein is also discussed. The paper concludes by reviewing elements of apparent universality between different bi-directionally moving cargos and by briefly considering possible reasons for the existence of bi-directional transport.

Animals↗
PubMed2003

Switching between microtubule- and actin-based transport systems in melanophores is controlled by cAMP levels.

BACKGROUND: Intracellular transport involves the movement of organelles along microtubules (MTs) or actin filaments (AFs) by means of opposite-polarity MT motors or actin-dependent motors of the myosin family. The correct delivery of organelles to their different destinations involves a precise coordination of the two transport systems. Such coordination could occur through regulation of the densities of the two cytoskeletal systems or through regulation of the activities of the cytoskeletal motors by signaling mechanisms. RESULTS: To investigate the mechanisms of switching between MT and AF-dependent transport, we examine the influence of the densities of the MT and AF network on pigment transport in fish melanophores. We also change signaling by using activators and inhibitors of Protein Kinase A (PKA). We find that the key parameters characterizing pigment granule transport along MTs do not depend on MT density and are not significantly altered by complete disruption of AFs. In contrast, the kinetics of changes in these parameters correlate with the kinetics of changes in the intracellular levels of cAMP and are affected by the inhibitors of PKA, suggesting the regulation of MT- and AF-dependent motors by cAMP-induced signaling. Furthermore, perturbation of cAMP levels prevents the transfer of pigment granules from MTs onto AFs. CONCLUSIONS: We conclude that the switching of pigment granules between the two major cytoskeletal systems is independent of the densities of MT or AF but is tightly controlled by signaling events.

Actin Cytoskeleton↗
PubMed2003

A determinant for directionality of organelle transport in Drosophila embryos.

BACKGROUND: Motor-driven transport along microtubules is a primary cellular mechanism for moving and positioning organelles. Many cargoes move bidirectionally by using both minus and plus end-directed motors. How such cargoes undergo controlled net transport is unresolved. RESULTS: Using a combination of genetics, molecular biology, and biophysics, we have identified Halo, a novel regulator of lipid droplet transport in early Drosophila embryos. In embryos lacking Halo, net transport of lipid droplets, but not that of other cargoes, is specifically altered; net transport is minus-end directed at developmental stages when it is normally plus-end directed. This reversal is due to an altered balance of motion at the level of individual organelles; without Halo, travel distances and stall forces are reduced for plus-end and increased for minus-end motion. During development, halo mRNA is highly upregulated just as net plus-end transport is initiated (phase II), and its levels drop precipitously shortly before transport becomes minus-end directed (phase III). Exogenously provided Halo prevents the switch to net minus-end transport in phase III in wild-type embryos and induces net plus-end transport during phase II in halo mutant embryos. This mechanism of regulation is likely to be of general importance because the Drosophila genome encodes a family of related proteins with similar sequences, each transiently expressed in distinct domains. CONCLUSIONS: We conclude that Halo acts as a directionality determinant for embryonic droplet transport and is the first member of a new class of transport regulators.

Amino Acid Sequence↗
PubMed2003

Dynactin: coordinating motors with opposite inclinations.

In eukaryotic cells, many organelles are transported bidirectionally along microtubules by kinesin and dynein. These opposite-polarity motors appear to be coordinated to avoid interfering with each other's function. New work has provided the first molecular insight into how such coordination might occur.

Animals↗
PubMed2002

Coordination of opposite-polarity microtubule motors.

Many cargoes move bidirectionally, frequently reversing course between plus- and minus-end microtubule travel. For such cargoes, the extent and importance of interactions between the opposite-polarity motors is unknown. In this paper we test whether opposite-polarity motors on lipid droplets in Drosophila embryos are coordinated and avoid interfering with each other's activity, or whether they engage in a tug of war. To this end we impaired the minus-end transport machinery using dynein and dynactin mutations, and then investigated whether plus-end motion was improved or disrupted. We observe a surprisingly severe impairment of plus-end motion due to these alterations of minus-end motor activity. These observations are consistent with a coordination hypothesis, but cannot be easily explained with a tug of war model. Our measurements indicate that dynactin plays a crucial role in the coordination of plus- and minus-end-directed motors. Specifically, we propose that dynactin enables dynein to participate efficiently in bidirectional transport, increasing its ability to stay "on" during minus-end motion and keeping it "off" during plus-end motion.

Animals↗
PubMed2002

Interactions and regulation of molecular motors in Xenopus melanophores.

Many cellular components are transported using a combination of the actin- and microtubule-based transport systems. However, how these two systems work together to allow well-regulated transport is not clearly understood. We investigate this question in the Xenopus melanophore model system, where three motors, kinesin II, cytoplasmic dynein, and myosin V, drive aggregation or dispersion of pigment organelles called melanosomes. During dispersion, myosin V functions as a "molecular ratchet" to increase outward transport by selectively terminating dynein-driven minus end runs. We show that there is a continual tug-of-war between the actin and microtubule transport systems, but the microtubule motors kinesin II and dynein are likely coordinated. Finally, we find that the transition from dispersion to aggregation increases dynein-mediated motion, decreases myosin V--mediated motion, and does not change kinesin II--dependent motion. Down-regulation of myosin V contributes to aggregation by impairing its ability to effectively compete with movement along microtubules.

Actin Cytoskeleton↗
PubMed2002

The importance of lattice defects in katanin-mediated microtubule severing in vitro.

The microtubule-severing enzyme katanin uses ATP hydrolysis to disrupt noncovalent bonds between tubulin dimers within the microtubule lattice. Although its microtubule severing activity is likely important for fundamental processes including mitosis and axonal outgrowth, its mechanism of action is poorly understood. To better understand this activity, an in vitro assay was developed to enable the real-time observation of katanin-mediated severing of individual, mechanically unconstrained microtubules. To interpret the experimental observations, a number of theoretical models were developed and compared quantitatively to the experimental data via Monte Carlo simulation. Models that assumed that katanin acts on a uniform microtubule lattice were incompatible with the in vitro data, whereas a model that assumed that katanin acts preferentially on spatially infrequent microtubule lattice defects was found to correctly predict the experimentally observed breaking rates, number and spatial frequency of severing events, final levels of severing, and sensitivity to katanin concentration over the range 6-300 nM. As a result of our analysis, we propose that defects in the microtubule lattice, which are known to exist but previously not known to have any biological function, serve as sites for katanin activity.

Adenosine Triphosphatases↗